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Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.

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Fast-time-lapse luminescence microscopy in budding yeast. Montage of LEU1pr-FLuc-yEVenus-PEST switching between leucine repression and induction. Our multicopy strain AMV167 was grown at 30°C on a CellAsic microfluidic device that was mounted on a DeltaVision microscope (Applied Precision, Issaquah, WA) within an incubation chamber. We imaged cells every 4 min. To capture full luminescence, each image is a sum projection of five z-stacks separated by 0.4 μm, with 10-s exposures for each stack for a subminute total. We also acquired a 2-ms fluorescence image, followed by a 7-ms phase image for image segmentation. The difference in exposure times indicates that yEVenus emits ∼104-fold more photons per second than FLuc, which is consistent with known photon flux outputs of fluorescent proteins and luciferases. (A) Previously induced cells were repressed during the switch from SCD-Leu to SCD+Leu medium at 390 min (vertical line in B). (B) Single-cell luminescence and fluorescence traces of repression (n = 16 cells). Image segmentation was done with CellStat program (Kvarnström et al., 2008). Rapid increases in signal are the new buds identified and tracked by the segmentation program. Best fit of a mathematical model of gene repression with delay to average luminescence signal (dark brown) and average fluorescence signal (dark green) is shown in black; see Materials and Methods and Supplemental Table S1 for details. Thin, black dotted lines are the 95% confidence interval of the best fit curve. The luminescence background is set by EMCCD camera noise, whereas fluorescence background is set by cellular autofluorescence. (C) Previously repressed cells were induced during the switch from SCD+Leu to SCD-Leu medium at 390 min (vertical line in D). (D) Single cell luminescence and fluorescence traces of induction (n = 18 cells). Scale bar, 10 μm.
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Figure 4: Fast-time-lapse luminescence microscopy in budding yeast. Montage of LEU1pr-FLuc-yEVenus-PEST switching between leucine repression and induction. Our multicopy strain AMV167 was grown at 30°C on a CellAsic microfluidic device that was mounted on a DeltaVision microscope (Applied Precision, Issaquah, WA) within an incubation chamber. We imaged cells every 4 min. To capture full luminescence, each image is a sum projection of five z-stacks separated by 0.4 μm, with 10-s exposures for each stack for a subminute total. We also acquired a 2-ms fluorescence image, followed by a 7-ms phase image for image segmentation. The difference in exposure times indicates that yEVenus emits ∼104-fold more photons per second than FLuc, which is consistent with known photon flux outputs of fluorescent proteins and luciferases. (A) Previously induced cells were repressed during the switch from SCD-Leu to SCD+Leu medium at 390 min (vertical line in B). (B) Single-cell luminescence and fluorescence traces of repression (n = 16 cells). Image segmentation was done with CellStat program (Kvarnström et al., 2008). Rapid increases in signal are the new buds identified and tracked by the segmentation program. Best fit of a mathematical model of gene repression with delay to average luminescence signal (dark brown) and average fluorescence signal (dark green) is shown in black; see Materials and Methods and Supplemental Table S1 for details. Thin, black dotted lines are the 95% confidence interval of the best fit curve. The luminescence background is set by EMCCD camera noise, whereas fluorescence background is set by cellular autofluorescence. (C) Previously repressed cells were induced during the switch from SCD+Leu to SCD-Leu medium at 390 min (vertical line in D). (D) Single cell luminescence and fluorescence traces of induction (n = 18 cells). Scale bar, 10 μm.

Mentions: We screened all multicopy transformants using a 96-well plate luminescence assay and selected the “brightest” strains (Figure 3B) for time-lapse luminescence microscopy. The variability in luminescence between transformants arises from unequal copy number integration in chromosomal loci due to homologous ends-in recombination. Time-lapse luminescence microscopy using standard, agarose pad methods initially exhibited bright luminescence signal. However, the signal disappeared within 15 min. We reasoned that luciferase substrates (d-luciferin, oxygen) were being depleted. Thus we combined a CellAsic microfluidic device (EMD Millipore, Billerica, MA) with time-lapse luminescence microscopy. The microfluidic device trapped yeast cells and maintained growth medium at constant levels through perfusion. The medium exchange time was <1 min, and cells were unable to deplete their substrates. With microfluidics, we consistently measured in vivo gene expression dynamics with subminute time resolution for >12 h (Figure 4). We only stopped time lapse because the yeast microcolony extended beyond the field of view. We further validated our method by benchmarking and quantifying gene induction and repression dynamics of many green, yellow, and red luciferase reporters for several metabolite-repressed yeast promoters (MET17, LEU1, ADE17, LYS9); see Supplemental Figures S2–S4 and Supplemental Table S1.


Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy.

Mazo-Vargas A, Park H, Aydin M, Buchler NE - Mol. Biol. Cell (2014)

Fast-time-lapse luminescence microscopy in budding yeast. Montage of LEU1pr-FLuc-yEVenus-PEST switching between leucine repression and induction. Our multicopy strain AMV167 was grown at 30°C on a CellAsic microfluidic device that was mounted on a DeltaVision microscope (Applied Precision, Issaquah, WA) within an incubation chamber. We imaged cells every 4 min. To capture full luminescence, each image is a sum projection of five z-stacks separated by 0.4 μm, with 10-s exposures for each stack for a subminute total. We also acquired a 2-ms fluorescence image, followed by a 7-ms phase image for image segmentation. The difference in exposure times indicates that yEVenus emits ∼104-fold more photons per second than FLuc, which is consistent with known photon flux outputs of fluorescent proteins and luciferases. (A) Previously induced cells were repressed during the switch from SCD-Leu to SCD+Leu medium at 390 min (vertical line in B). (B) Single-cell luminescence and fluorescence traces of repression (n = 16 cells). Image segmentation was done with CellStat program (Kvarnström et al., 2008). Rapid increases in signal are the new buds identified and tracked by the segmentation program. Best fit of a mathematical model of gene repression with delay to average luminescence signal (dark brown) and average fluorescence signal (dark green) is shown in black; see Materials and Methods and Supplemental Table S1 for details. Thin, black dotted lines are the 95% confidence interval of the best fit curve. The luminescence background is set by EMCCD camera noise, whereas fluorescence background is set by cellular autofluorescence. (C) Previously repressed cells were induced during the switch from SCD+Leu to SCD-Leu medium at 390 min (vertical line in D). (D) Single cell luminescence and fluorescence traces of induction (n = 18 cells). Scale bar, 10 μm.
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Figure 4: Fast-time-lapse luminescence microscopy in budding yeast. Montage of LEU1pr-FLuc-yEVenus-PEST switching between leucine repression and induction. Our multicopy strain AMV167 was grown at 30°C on a CellAsic microfluidic device that was mounted on a DeltaVision microscope (Applied Precision, Issaquah, WA) within an incubation chamber. We imaged cells every 4 min. To capture full luminescence, each image is a sum projection of five z-stacks separated by 0.4 μm, with 10-s exposures for each stack for a subminute total. We also acquired a 2-ms fluorescence image, followed by a 7-ms phase image for image segmentation. The difference in exposure times indicates that yEVenus emits ∼104-fold more photons per second than FLuc, which is consistent with known photon flux outputs of fluorescent proteins and luciferases. (A) Previously induced cells were repressed during the switch from SCD-Leu to SCD+Leu medium at 390 min (vertical line in B). (B) Single-cell luminescence and fluorescence traces of repression (n = 16 cells). Image segmentation was done with CellStat program (Kvarnström et al., 2008). Rapid increases in signal are the new buds identified and tracked by the segmentation program. Best fit of a mathematical model of gene repression with delay to average luminescence signal (dark brown) and average fluorescence signal (dark green) is shown in black; see Materials and Methods and Supplemental Table S1 for details. Thin, black dotted lines are the 95% confidence interval of the best fit curve. The luminescence background is set by EMCCD camera noise, whereas fluorescence background is set by cellular autofluorescence. (C) Previously repressed cells were induced during the switch from SCD+Leu to SCD-Leu medium at 390 min (vertical line in D). (D) Single cell luminescence and fluorescence traces of induction (n = 18 cells). Scale bar, 10 μm.
Mentions: We screened all multicopy transformants using a 96-well plate luminescence assay and selected the “brightest” strains (Figure 3B) for time-lapse luminescence microscopy. The variability in luminescence between transformants arises from unequal copy number integration in chromosomal loci due to homologous ends-in recombination. Time-lapse luminescence microscopy using standard, agarose pad methods initially exhibited bright luminescence signal. However, the signal disappeared within 15 min. We reasoned that luciferase substrates (d-luciferin, oxygen) were being depleted. Thus we combined a CellAsic microfluidic device (EMD Millipore, Billerica, MA) with time-lapse luminescence microscopy. The microfluidic device trapped yeast cells and maintained growth medium at constant levels through perfusion. The medium exchange time was <1 min, and cells were unable to deplete their substrates. With microfluidics, we consistently measured in vivo gene expression dynamics with subminute time resolution for >12 h (Figure 4). We only stopped time lapse because the yeast microcolony extended beyond the field of view. We further validated our method by benchmarking and quantifying gene induction and repression dynamics of many green, yellow, and red luciferase reporters for several metabolite-repressed yeast promoters (MET17, LEU1, ADE17, LYS9); see Supplemental Figures S2–S4 and Supplemental Table S1.

Bottom Line: The photon flux per luciferase is significantly lower than that for fluorescent proteins.Fluorescence of an optimized reporter (Venus) lagged luminescence by 15-20 min, which is consistent with its known rate of chromophore maturation in yeast.Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genome Sciences and Policy, Duke University, Durham, NC 27710 Duke Center for Systems Biology, Duke University, Durham, NC 27710 Department of Biology, Duke University, Durham, NC 27710.

Show MeSH
Related in: MedlinePlus